Throughout this application, various publications are referenced by author and date. Full citations for these publications may be found listed alphabetically near the end of the specification. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein.
Beclin is a novel, 61 kd, Bcl-2-interacting, coiled coiled protein encoded by a gene located on a tumor susceptibility locus on chromosome 17q21. This application shows that Beclin has tumor suppressor and anti-apoptotic functions. The region of chromosome 17q21 encoding Beclin is deleted in 50-75% of cases of prostate cancer, ovarian cancer, and breast cancer.
This application shows that beclin is a tumor suppressor gene important for breast, ovarian, and prostate cancer. The experiments herein show the presence of aberrant splice products in sporadic epithelial ovarian cancers that contain monoallelic deletions of beclin, and the loss of Beclin protein expression in sporadic invasive breast carcinomas. These findings suggest that mutations in beclin splice site junctions, mutations in regulatory regions of the beclin gene (e.g. promoters, enhancers, 5xe2x80x2 and 3xe2x80x2 nontranslated regions), mutations in the coding region which may lead to decreased protein stability, and/or hypermethylation of the promoter region, are important mechanisms for functional inactivation of beclin and subsequent tumorigenesis in breast, ovarian, and/or prostate cancer.
Monoallelic deletions of chromosome 17q21, detected by loss-of heterozygosity (LOH), are found in 50% of breast carcinomas, 50-75% of ovarian carcinomas, and 40% of prostate carcinomas (Cropp et al. 1993; Futreal et al. 1992; Saito et al. 1993; Eccles et al 1992; Russell et al. 1990; Sato et al. 1991; Yang-Feng et al. 1993; Gao et al. 1995; Gao et al. 1995). This frequent LOH in chromosome 17q21. suggests that inactivation of one or more tumor suppressor genes located on this chromosomal arm are important for breast ovarian, and prostate carcinogenesis. In 1994, the existence of one such gene, BRCA1, was identified by positional cloning, and mutations in BRCA1 are now accepted to be an important cause of hereditary breast and ovarian cancer (Futreal et al. 1994; Miki et al. 1994). However, it remains unclear whether functional inactivation of BRCA1 represents an important molecular event in the development of sporadic breast and ovarian carcinomas. (Vogelstein and Kinzler 1994; Tangir et al. 1994). To fulfill the Knudson two-hit hypothesis (Knudson 1985) for inactivation of a tumor suppressor gene as a mechanism for oncogenesis, a mutation has to be found in the remaining allele of tumors showing BRCA1 locus deletion. To date, somatic mutations in BRCA1 have been found in very few cases of sporadic cancers (Futreal et al. 1994; Hosking et al. 1995; Merajver et al. 1995; Takahashi et al. 1995).
In further support of this hypothesis, more finely detailed deletion mapping has identified a 400 kb deletion unit approximately 60 kb centromeric to the BRCA gene in sporadic epithelial ovarian cancer (Tangir et al. 1996). This deletion unit may contain a putative tumor suppressor important for sporadic ovarian cancer and contains approximately 12 genes which were identified during the search for BRCA1. Six of them are known genes or human homologs of other species; gamma tubulin, homolog of D. melanogaster enhancer of zeste, pseudogene of HMG17, homology of Pacific electric ray VAT1, glucose-6-phosphatase and Ki antigen and six of them are novel genes.
This application describes the identification of the full-length open reading frame and functional characterization of one of these six novel genes, which is herein referred to as xe2x80x9cbeclinxe2x80x9d. This application shows that Beclin overexpression inhibits cellular proliferation in vitro and suppresses the ability of human breast carcinoma cells to form colonies in soft agar and tumors in nude mice. Thus, these studies demonstrate that beclin has tumor suppressor function. This application describes the sequences of the 5xe2x80x2 and 3xe2x80x2 untranslated regions of beclin, and provides evidence that, beclin may be functionally inactivated in human cancers by mutations in the gene which lead to loss of Beclin protein expression or by splice site mutations which lead to aberrant beclin splicing.
The cellular antiapoptotic gene bcl-2 represents a novel class of antiviral host defense molecules which function both by restricting viral replication and by preventing virus-induced cell death. Bcl-2 blocks apoptosis in vitro induced by several different RNA viruses, including Sindbis virus, influenza virus, reovirus, Semliki Forest virus, LaCrosse virus, and Japanese B encephalitis virus. Previously, we have shown that Bcl-2 overexpression in virally infected neurons in vivo also protects mice against fatal encephalitis caused by the prototypic alphavirus, Sindbis virus. The protective effects of Bcl-2 against fatal Sindbis virus encephalitis were associated with a reduction both in neuronal apoptotic death and in central nervous system (CNS) viral replication. A similar antiviral effect of Bcl-2 overexpression has been observed during Sindbis virus infection in cultured AT3 cells as well as during influenza virus infection of MDCK cells, Japanese B encephalitis virus infection of N18 cells, and Semliki Forest virus infection of AT3 cells. Although the role of endogenous Bcl-2 in antiviral defense has yet to be evaluated, these studies support the hypothesis that Bcl-2 may be important in protecting cells against viral infections.
The process of autophagy, or bulk degradation of cellular proteins through an autophagosomic-lysosomal pathway, is important in normal growth control and may be defective in tumor cells. However, little is known about the genetic mediators of autophagy in mammalian cells and whether genetic mediators of autophagy influence tumor development. Recently, 14 genes, the apg genes, have been identified in S. cerevisiae that are required for yeast autophagy1. The mammalian gene encoding Beclin 12, a novel Bcl-2-interacting, coiled-coil protein, shares structural similarity with the yeast autophagy gene product, Apg6/Vps30p3,4, and is monoallelically deleted in 40-75% of sporadic human breast cancers and ovarian cancers 5-11. Here we show, using gene transfer techniques, that beclin 1 promotes autophagy both in autophagy-defective yeast with a targeted disruption of apg6/vps30 and in autophagy-defective human MCF7 breast carcinoma cells. The autophagy-promoting activity of beclin 1 in MCF7 cells is associated with an ability to inhibit MCF7 cellular proliferation, in vitro clonigenicity, and tumorigenesis in nude mice. Furthermore, endogenous Beclin 1 protein expression is frequently undetectable or low in malignant human breast epithelial cell lines and tissue, but is expressed ubiquitously at high levels in normal breast epithelia. Thus, beclin 1 is a mammalian autophagy gene with tumor suppressor function that is expressed at decreased levels in human breast cancer. These findings indicate that there is an evolutionarily conserved genetic link between autophagy and tumor suppressor pathways, and raise the possibility that decreased expression of autophagy proteins may contribute to the development or progression of breast arid other human malignancies.
Autophagy is an evolutionarily conserved process that occurs in both yeast and mammalian cells in which there is bulk degradation of cellular contents via an autotphagosomal-lysosomal pathway. The process of autophagy liberates free amino acids and nucleotides and enable cells to survive under nutrient deprivation conditions as well as to undergo structural remodeling during differentiation. Protein degradation through an autophagy pathway has also been postulated to serve as a mechanism for negative regulation of cell growth. In support of this theory, cancerous transformation in vitro is associated with decreased rates of autophagic degradation. Malignant cell lines display less autophagic activity than their normal counterparts and are less prone to increase autophagic activity in response to serum deprivation or high cell density. Furthermore, in rat liver carcinogenesis models, there is an inverse correlation between stage of malignancy and autophagic activity. However, it is not known whether reduced autophagy in tumor cells directly contributes to deregulated cell growth, or alternatively, is the result of malignant transformation.
This invention provides a method of diagnosing a predisposition to carcinoma in a subject comprising: a) obtaining a nucleic acid sample from the subject; and b) determining whether one or more of the subject""s beclin alleles or regulatory regions to those alleles are deleted or different from the wild type so as to reduce the subject""s expression of polypeptide having tumor suppressor activity.
This invention also provides the above-described method, wherein one or more beclin alleles contain splice site junction mutations.
This invention provides also the above-described methods, wherein the determining of step (b) comprises determining whether one or more beclin promoter regions are hypermethylated.
Further, this invention provides the above-described methods, wherein the determining of step (b) comprises determining whether one or more beclin enhancer regions differ from the wild type.
This invention further provides the above-described methods, wherein the determining of step (b) comprises determining whether one or more beclin 5xe2x80x2 or 3xe2x80x2 nontranslated regions differ from the wild type.
This invention also provides the above-described methods, wherein the determining of step (b) comprises determining whether one or more beclin alleles have a premature termination codon.
This invention also provides the above-described methods, wherein a majority of the nucleotides corresponding to positions 1042-1185 of FIG. 1B have been deleted.
Further, this invention provides the above-described methods, wherein the subject was preselected based upon an abnormality at chromosome position 17q21.
This invention also provides the above-described methods, wherein the nucleic acid sample is obtained from the subject""s blood.
This invention further provides a method of diagnosing a predisposition to carcinoma in a subject, comprising determining whether the subject has an abnormal subcellular distribution of Beclin.
In addition, this invention provides a method of diagnosing a predisposition to carcinoma in a subject, comprising contacting the contents of one or more of the subject""s cells with one or more antibodies or fragments thereof specific to Beclin.
This invention provides a method for inhibiting viral replication comprising contacting an effective amount of Beclin with the virus infected cell, thereby inhibiting the viral replication where the method for inhibiting viral replication comprising contacting induction of the expression of Beclin with the virus infected cell, thereby inhibiting the viral replication.
A method of treating a viral infection in a subject, wherein the viral infection is alleviated by the expression of Beclin which comprises administering to the subject an effective amount of the pharmaceutical composition effective to express Beclin, thereby treating the viral infection in the subject.
In addition, this invention provides a method for treating cancer comprising inducing increased expression of Beclin, where the method for treating cancer comprises administering to the subject a therapeutically effective amount of beclin son as to restore cell growth control.
This invention provides a pharmaceutical composition which comprises Beclin and a pharmaceutically acceptable carrier.